Increasing responses to checkpoint inhibitors by extracorporeal apheresis

ABSTRACT

The invention provides means, methods, and compositions of matter useful for enhancing tumor response to checkpoint inhibitors. In one embodiment, the invention teaches utilization of extracorporeal apheresis, specifically removal of various tumor derived, or tumor microenvironment derived immunological “blocking factors”. In one embodiment the invention provides the removal of soluble TNF-alpha receptors (sTNF-Rs) as a means of augmenting efficacy of immune checkpoint inhibitors. In one specific embodiment removal of sTNF-Rs is utilized to enhance efficacy of inhibitors of the PD-1/PD-L1 pathway, and/or the CD28/CTLA-4 pathway.

BACKGROUND

There is an increasing prevalence of cancer in human and its significantcontribution to mortality means there is a continuing need for newtherapies. Elimination of the cancer, a reduction in its size, thedisruption of its supporting vasculature, or reducing the number ofcancer cells circulating in the blood or lymph systems are goals ofcurrent cancer therapies. Mechanistically, therapies for cancer aredesigned to combat tumors or cells metastasizing from tumors typicallyrely on a cytotoxic activity. That activity might he a cytotoxic effectan active agent has itself or it might he an effect employed indirectlyby the active agent such as through the modulation of immune responses.

It is known that genetic and epigenetic changes occur in tissues as theytransform to take on the phenotype of a cancer cell. Different steps inthe malignant transformation process, including acquisition of themutator phenotype, which is associated with loss of tumor suppressoractivity, often results in the generation of neoantigens, which aresubject to immune recognition.

Various attempts have been made to help the immune system to fighttumors. One early approach involved a general stimulation of the immunesystem through the administration of bacteria (live or killed) to elicita general immune response which would also be directed against thetumor. Existing innate stimulators of immunity include BCG [1-10],lyophilized incubation mixture of group A Streptococcus pyogenes(OK-432) [11-26], CSF-470 [27, 28], as well as doses of chemotherapiesthat can selectively suppress Treg cells [29-39].

As far back as 1975 [40], it was known that administration ofnon-specific immune activators could induce local and in some casessystemic regression of cancer. For example, in one study, 6 patientswith intradermal metastases of malignant melanoma were treated withintralesional BCG. Four patients showed a good response with regressionof injected, and in some cases, uninjected lesions, whereas the othertwo developed metastatic visceral disease and died. Three of the sixpatients had complete regression of all lesions, and one exhibitedcomplete regression of untreated lesions. All remain free of disease.The fourth patient had complete regression of injected and of someuntreated lesions, but developed widespread dissemination and died.Three of four responders (i.e. those patients in whom treated lesionsdecreased in size by more than 50% for more than 1 month) showed adramatic increase in lymphocyte stimulation to melanoma antigens. Allresponders (four out of four) had a marked increase tophytohemagglutinin (PHA), whereas non responders had no increase inlymphocyte stimulation either to melanoma antigens or PHA. These datasuggested the other important point, which is that innate immuneactivation can lead to stimulation of antigen-specific T cell and B cellmediated immune responses. Recent approaches aimed at helping the immunesystem specifically to recognize tumor-specific antigens involveimmunization with cancer-specific antigens, typically combined with anadjuvant (a substance which is known to cause or enhance an immuneresponse) to the subject. Tumor specific antigens are well known andinclude the group of cancer testes antigens (CT antigens) or germ cellantigens that are reactivated in cancerous tissues. It is known that theusual lack of a powerful immune response to tumor associated antigens(TAAs) is due to a combination of factors. T cells have a key role inthe immune response, which is mediated through antigen recognition bythe T cell receptor (TCR), and they coordinate a balance betweenco-stimulatory and inhibitory signals known as immune checkpoints. Theseinhibitory signals function as natural suppressors of the immune systemas an important mechanism for for maintenance of self-tolerance and toprotect tissues from damage when the immune system is responding topathogenic infection. However, disregulated immune suppression reduceswhat could otherwise be a helpful response by the body to avoid thedevelopment of tumors. Cytokines, other stimulatory molecules such asCpG (stimulating dendritic cells), Toll-like receptor ligands and othermolecular adjuvants enhance the immune response. Co-stimulatoryinteractions involving T cells directly can be enhanced using agonisticantibodies to receptors including OX40, CD28, CD27 and CD137. Otherimmune system activating therapies include blocking and/or depletinginhibitory cells or molecules and include the use of antagonisticantibodies against what are known as immune checkpoints [41]. It isknown that immune cells express proteins that are immune checkpointsthat control and down-regulate the immune response. These are bestdefined in T lymphocytes and include PD-1, CTLA-4, TIM-3 and LAGS. Tumorcells express the ligands to these receptors. When T cells bind theligand to these proteins on the tumor cells, the T cell is turned offand does not attempt to attack the tumor cell. Thus, checkpoint immunesuppression is part of the complex strategy used by the tumor to evadethe patient's immune system and Is responsible for resistance toimmunotherapy. Biopharmaceutical companies have successfully developedtherapeutic checkpoint inhibitors that block the receptor/ligandinteraction to promote the adaptive immune response to the tumor. Sixcheckpoint inhibitors are currently approved, pembrolizumab, nivolumab,atezolizumab, avelumab, durvalumab, and ipilimumab for a wide variety ofsolid tumors including melanoma, lung, bladder, gastric cancers andothers. T cells are central to the immune response to cancers and thereis interest in the field in using tumor infiltrating lymphocytes (TILs)in the treatment and understanding of cancer. Through their T cellreceptors (TCRs), T cells are reactive to specific antigens within atumor. Tumor cells carry genetic mutations, many of which contributedirectly or indirectly to malignancy. A mutation in an expressedsequence will typically result in a neoantigen, an antigen that is notknown to the immune system and thus recognized as foreign and able toelicit an immune response. The importance of TIL is that they areassociated with superior patient prognosis including in gastric cancer[42], breast cancer [43-46], melanoma [47], head and neck cancer [48],thus suggesting an active role of the immune system in contributing tocancer survival.

Unfortunately, despite the great advances in understanding of theimmune-cancer interaction, and development of novel first in class drugsaround this concept, many patients still do not respond toimmunotherapies, and in some cases, those that respond suffer fromrelapse. The current invention teaches means of augmenting efficacy ofimmunotherapies, specifically of checkpoint inhibitors, by removal oftumor derived and/or tumor microenvironment derived immunologicalblocking factors using extracorporeal means.

SUMMARY

Embodiments herein are directed to methods of enhancing the efficacy ofan immune checkpoint inhibitor administered to a patient suffering froma tumor comprising: identifying a patient suffering from a tumor;administering an immunological checkpoint inhibitor to said patient totreat said tumor or ameliorate the effects of said tumor;extracorporeally removing immunological blocking factors that inhibitthe effectiveness of said immunological checkpoint inhibitor in anamount sufficient to augment the efficacy of said immunologicalcheckpoint inhibitor in either treating or ameliorating the effects ofsaid tumor, wherein said extracorporeal removal is conducted at a timeselected from the group consisting of: before, concurrently, andsubsequent to the administration of said immunological checkpointinhibitor.

More specifically, disclosed herein are methods wherein said efficacy ofsaid checkpoint inhibitor is based on an endpoint selected from thegroup consisting of: a) tumor regression; b) tumor stabilization; c)reduction in tumor growth; d) inhibition of metastasis; e) stabilizationof metastasis; f) reduction of metastatic growth; g) encapsulation oftumor and/or metastasis; h) augmentation of cytokines associated withtumor inhibition; i) decrease in cytokines associated with tumorprogression; j) suppression of angiogenesis; k) augmentation of tumorinfiltrating lymphocytes; 1) switch of intratumoral macrophages from M2to M1 phenotype; m) augmentation of tumor infiltrating dendritic cells;n) augmentation of tumor infiltrating killer T-cells o) reduction oftumor associated T regulatory cells; and p) reduction in tumorassociated myeloid suppressor cells.

According to further embodiments, said checkpoint inhibitor is an agentcapable of suppressing activity of a molecule selected from the groupconsisting of: PD-1, PD-L1, CTLA-4, PD-L2, LAG3, Tim3, 2B4, A2aR, ID02,B7-H3, B7-H4, BTLA, CD2, CD20, CD27, CD28, CD30, CD33, CD40, CD52, CD70,CD112, CD137, CD160, CD226, CD276, DR3, OX-40, GAL9, GITR, ICOS, HVEM,IDO1, KIR, LAIR, LIGHT, MARCO, PS, SLAM, TIGIT, VISTA, and VTCN1

According to other embodiments said immunological blocking factor issoluble TNF-alpha receptor.

According to further embodiments, disclosed herein are methods whereinsaid immunological blocking factor is selected from the group consistingof: a) soluble HLA-G; b) soluble MICA; c) interleukin-10; d)interleukin-20; e) VEGF; f) soluble IL-2 receptor; g) soluble IL-15receptor; h) interleukin-35 and i) soluble interferon gamma receptor.

According to more specific embodiments, said removal of solubleTNF-alpha receptor is performed by affinity capture to TNF-alphatrimers.

According to further embodiments said checkpoint inhibitor isadministered via a route selected from the group consisting of:intravenously, intramuscularly, parenterally, nasally, intratumorally,intraosseously, subcutaneously, sublingually, intrarectally,intrathecally, intraventricularly, orally, intraocularly, topically, orvia inhalation, nanocell and/or nanobubble injection.

According to more specific embodiments, the immunological checkpointinhibitor is selected from the group consisting of PD-1, PD-L1, andCTLA-4.

According to other embodiments, the inhibitor of PD-1 is an anti-PD-1antibody selected from the group consisting of nivolumab andpembrolizumab.

Further embodiment are directed to methods wherein the inhibitor ofPD-L1 is anti-PD-L1 antibody selected from the group consisting of:BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736,MSB0010718C, and MDX1105-01.

According to other embodiments, the inhibitor of CTLA-4 is ananti-CTLA-4 antibody selected from the group consisting of ipilimumaband tremelimumab.

According to certain embodiments, said removal of said soluble TNF-alphareceptor is performed using an extracorporeal affinity capture substratecomprising immobilized TNF-alpha molecules selected from the groupconsisting of: TNF-alpha trimers, native TNF-alpha molecules, andmutated forms of TNF-alpha, wherein said immobilized TNF-alpha moleculeson the extracorporeal affinity capture substrate have at least onebinding site capable of selectively binding to soluble TNF alphareceptor from a biological fluid.

Further methods include embodiments wherein said removal ofimmunological blocking factors is performed using an apheresis systemutilizing centrifugal plasma separation.

Additional methods include embodiments, wherein said removal ofimmunological blocking factors is performed using an apheresis systemutilizing membrane plasma separation.

Other aspects embody methods wherein enhancing efficacy of an immunecheckpoint inhibitor is accomplished by performing one or more clinicalprocedures involving the removal of tumor derived blocking factors toprepare and/or condition the patient.

Still further embodiments include methods wherein said removal ofsoluble TNF-alpha receptor is performed by affinity capture to TNF-alphatrimers.

According to more specific embodiments said checkpoint inhibitor isadministered intravenously, intramuscularly, parenterally, nasally,intratumorally, intraosseously, subcutaneously, sublingually,intrarectally, intrathecally, intraventricularly, orally, topically, orvia inhalation, nanocell and/or nanobubble injection.

According to further embodiments said extracorporeal removal of immuneblocking factors primes antigen presenting cells for enhanced ability toproduce interleukin-12 subsequent to administration of a checkpointinhibitor.

Further embodiments are directed to an immune checkpoint inhibitor foruse in a method of treating a tumor in a patient, the method comprising:identifying a patient suffering from a tumor; administering animmunological checkpoint inhibitor to said patient to treat said tumoror ameliorate the effects of said tumor; and extracorporeally removingimmunological blocking factors that inhibit the effectiveness of saidimmunological checkpoint inhibitor, wherein said extracorporeal removalis conducted at a time selected from the group consisting of: before,concurrently, and subsequent to the administration of said immunologicalcheckpoint inhibitor. All methods disclosed herein can be used with saidimmune checkpoint inhibitors.

DESCRIPTION OF THE INVENTION

The invention discloses means of augmenting therapeutic ability ofimmunological checkpoint inhibitors by removal of immunological blockingfactors through extracorporeal means. In one embodiment, the inventionrelates to the field of cancer therapy, specifically means of augmentingefficacy of cancer therapy. In particular, the invention providesmethods of generating T cell populations capable of promoting thesuppression of cancer, as well as directly killing the cancer. As usedherein, the term “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects include preventingoccurrence or recurrence of disease, alleviation of symptoms, anddiminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, lowering the rate of diseaseprogression, amelioration or palliation of the disease state, andinducing remission or improving prognosis.

The term “extracorporeal means” is defined as the use of anextracorporeal device or system through which blood or bloodconstituents obtained from a patient are passed through a device for theremoval of the immune inhibitor(s) and wherein the blood or bloodconstituents that are depleted of immune inhibitor(s) are reinfused intothe patient. The extracorporeal device is comprised of materials thatselectively binds to and captures the specified inhibitor(s) to preventthem from being reinfused into the patient.

The term “affinity capture” means the selective binding of a specificsubstance or molecule by a chemical attraction.

The term “affinity capture substrate” means a material comprising anaffinity capture molecule.

The term “antibody” includes therapeutic antibodies suitable fortreating patients; such as abagovomab, adecatumumab, afutuzumab,alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab,bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab,brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab,cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab,duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab,ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab,farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab,ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab,igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab,iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab,lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab,mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab,nimotuzumab, nofetumomabn, ocaratuzumab, ofatumumab, olaratumab,onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab,patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab,radretumab, rilotumumab, rituximab, robatumumab, satumomab,sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab,taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tocilizumab,tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab,vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8. In some embodimentsthe invention teaches the combination use of extracorporeal removal ofblocking factors as a means of augmenting therapeutic efficacy of thementioned antibodies. In other embodiments the combination ofantibodies, together with extracorporeal removal of blocking factors, isfurther combined with administration of checkpoint inhibitors. Theinvention is particularly of importance to therapeutic antibodies whoseactions are mediated by antibody dependent cellular toxicity. Thementioned antibodies can be utilized individually or in combination.Furthermore administration of other immune modulators is envisionedwithin the scope of the invention. Immune modulators may be activatorsof innate immunity such as toll like receptor agonists. Other immunemodulators stimulate adaptive immunity such as T and B cells.Furthermore, immune stimulation by be achieved by removal of immunesuppressive cells utilizing approaches that deplete myeloid derivedsuppressor cells, Th3 cells, T regulatory cells, type 2 neutrophils,type 2 macrophages and eosinophils.

The terms “antigen-presenting cell (s)”, “APC” or “APCs” include bothintact, whole cells as well as other molecules (all of allogeneicorigin) which are capable of inducing the presentation of one or moreantigens, preferably in association with class I MHC molecules, and alltypes of mononuclear cells which are capable of inducing an allogeneicimmune response. Preferably whole viable cells are used as APCs.Examples of suitable APCs are, but not limited to, whole cells such asmonocytes, macrophages, DCs, monocyte-derived DCs, macrophage-derivedDCs, B cells and myeloid leukemia cells e. g. cell lines THP-1, U937,HL-60 or CEM-CM3. Myeloid leukemia cells are said to provide so calledpre-monocytes. In some embodiments of the invention, tumor inducedimmaturity of antigen presenting cells is overcome by extracorporealremoval of tumor associated blocking factors. Said removal results in apredisposition of antigen presenting cells to mature in response toadministration of checkpoint inhibitors.

The terms “cancer”, “neoplasm” and “tumor” are used interchangeably andin either the singular or plural form, as appearing in the presentspecification and claims, refer to cells that have undergone a malignanttransformation that makes them pathological to the host organism.Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e. g. by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Non-limitingexamples of tumors/cancers relevant for the present invention arecarcinomas (e.g. breast cancer, prostate cancer, lung cancer, colorectalcancer, renal cancer, gastric cancer and pancreatic cancer), sarcomas(e.g. bone cancer and synovial cancer), neuro-endocrine tumors (e.g.glioblastoma, medulloblastoma and neuroblastoma), leukemias, lymphomasand squamous cell cancer (e.g. cervical cancer, vaginal cancer and oralcancer). Further, non-limiting examples of tumors/cancers relevant forthe present invention are, glioma, fibroblastoma, neurosarcoma, uterinecancer, melanoma, testicular tumors, astrocytoma, ectopichormone-producing tumor, ovarian cancer, bladder cancer, Wilm's tumor,vasoactive intestinal peptide secreting tumors, head and neck squamouscell cancer, esophageal cancer, or metastatic cancer. Prostate cancerand breast cancer are particularly preferred.

For the practice of the invention, the term “chemotherapy” is meant toencompass any non-proteinaceous (i.e, non-peptidic) chemical compounduseful in the treatment of cancer. Examples of chemotherapeutic agentsinclude reactive oxygen agents such as artimesinin and alkylating agentssuch as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding alfretamine, triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimemylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingsynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (articularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, foremustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammaII and calicheamicin phiII,see, e.g., Agnew, Chem. Intl. Ed. Engl, 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adramycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g., paclitaxel (TAXOL™, Bristol Meyers Squibb Oncology, Princeton,N.J.) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (GEMZAR™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vancristine; vinorelbine (NAVELBINE™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil,leucovorin, and irinotecan) and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In some embodiments the efficacy ofchemotherapy is augmented by utilization of extracorporeal removal ofblocking factors. Furthermore, combination of the mentionedchemotherapies may be utilized together with checkpoint inhibitors. Theinvention is particularly relevant in situations where efficacy ofchemotherapy is related to immunological activity.

The terms “extracorporeal system” and “extracorporeal removal” refer toone or methods for depleting concentrations of substances from wholeblood and/or plasma, wherein said substances are immune suppressive.Methods for depleting substances from plasma can utilize systems thatperform plasma separation using centrifugal force or separation viamembrane, including but not limited to tangential flow systems and/orcapillary means. In one embodiment, said extracorporeal means is asingle-chain TNF-alpha based affinity column, termed the “LW-02” device,which may be used in combination with the Terumo Optia apheresis system.

The term “myeloid suppressor cell” is equivalent to immature myeloidprogenitor cells, myeloid derived suppressor cells, natural suppressorcells, or immature neutrophil/monocyte precursors.

The terms “vaccine”, “immunogen”, or immunogenic composition” are usedherein to refer to a compound or composition that is capable ofconferring a degree of specific immunity when administered to a human oranimal subject. As used in this disclosure, a “cellular vaccine” or“cellular immunogen” refers to a composition comprising at least onecell population, which is optionally inactivated, as an activeingredient. The immunogens, and immunogenic compositions of thisinvention are active, which mean that they are capable of stimulating aspecific immunological response (such as an anti-tumor antigen oranti-cancer cell response) mediated at least in part by the immunesystem of the host. The immunological response may comprise antibodies,immunoreactive cells (such as helper/inducer or cytotoxic cells), or anycombination thereof, and is preferably directed towards an antigen thatis present on a tumor towards which the treatment is directed. Theresponse may be elicited or re-stimulated in a subject by administrationof either single or multiple doses. A compound or composition is“immunogenic” if it is capable of either: a) generating an immuneresponse against an antigen (such as a tumor antigen) in a naiveindividual; or b) reconstituting, boosting, or maintaining an immuneresponse in an individual beyond what would occur if the compound orcomposition was not administered. A composition is immunogenic if it iscapable of attaining either of these criteria when administered insingle or multiple doses.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12 amino acids or evenlonger, and in case of MHC class II peptides (e.g. elongated variants ofthe peptides of the invention) they can be as long as 15, 16, 17, 18,19, 20 or 23 or more amino acids in length. Furthermore, the term“peptide” shall include salts of a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. Preferably,the salts are pharmaceutical acceptable salts of the peptides, such as,for example, the chloride or acetate (trifluoro-acetate) salts. It hasto be noted that the salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides are not salts in vivo. The term “peptide” shallalso include “oligopeptide”. The term “oligopeptide” is used herein todesignate a series of amino acid residues, connected one to the othertypically by peptide bonds between the alpha-amino and carbonyl groupsof the adjacent amino acids. The length of the oligopeptide is notcritical to the invention, as long as the correct epitope or epitopesare maintained therein. The oligopeptides are typically less than about30 amino acid residues in length, and greater than about 15 amino acidsin length.

The human in need thereof may be an individual who has or is suspectedof having a cancer. In some of variations, the human is at risk ofdeveloping a cancer (e.g., a human who is genetically or otherwisepredisposed to developing a cancer) and who has or has not beendiagnosed with the cancer. As used herein, an “at risk” subject is asubject who is at risk of developing cancer (e.g., a hematologicmalignancy). The subject may or may not have detectable disease, and mayor may not have displayed detectable disease prior to the treatmentmethods described herein. An at-risk subject may have one or moreso-called risk factors, which are measurable parameters that correlatewith development of cancer, such as described herein. A subject havingone or more of these risk factors has a higher probability of developingcancer than an individual without these risk factor(s). These riskfactors may include, for example, age, sex, race, diet, history ofprevious disease, presence of precursor disease, genetic (e.g.,hereditary) considerations, and environmental exposure. In someembodiments, a human at risk for cancer includes, for example, a humanwhose relatives have experienced this disease, and those whose risk isdetermined by analysis of genetic or biochemical markers. Prior historyof having cancer may also be a risk factor for instances of cancerrecurrence. In some embodiments, provided herein is a method fortreating a human who exhibits one or more symptoms associated withcancer (e.g., a hematologic malignancy). In some embodiments, the humanis at an early stage of cancer. In other embodiments, the human is at anadvanced stage of cancer.

Overall survival (OS) is defined as the time elapsed from start oftreatment until death of any cause. Progression Free Survival (PFS)(RECIST 1.1) is calculated from start of treatment until diseaseprogression or death. Objective response rate [CR (Complete Response) orPR (Partial Response) or SD (Stable Disease)] is defined as the percentof patients with best confirmed response CR or PR or SD, using CT orMRI, and determined by a central reader per RECIST 1.1. The responsemust be confirmed by a subsequent determination greater than or equal to4 weeks apart. In some instances PET is used. The evaluations andmeasurements are performed at screening, then at 8-week intervalsstarting from first treatment until PD (Progressive Disease) orinitiation of another or additional anti-tumor therapy, whichever occursfirst. In addition, scans are performed at each long-term follow-upvisit until progression.

In one embodiment of the invention patients are chosen in which a highdegree of cancer associated immune suppression present. Immunesuppression is assessed using different means known in the art and caninclude quantification of number of immune cells in circulation,quantification of activity of immune cells in circulation,quantification of the number of immune cells found intratumorally,quantification of activity of immune cells found intratumorally,quantification of the number of immune cells found peritumorally, andquantification of activity of immune cells found peritumorally. In someembodiments of the invention, quantification of immune cells comprisesidentification and assessment of activity of cells possessing tumorcytolytic and/or tumor inhibitory activity, such cells include naturalkiller cells (NK), gamma delta T cells, natural killer T cells (NKT),innate lymphoid cells, cytotoxic T lymphocytes (CTL), and helper T cells(Th). Activities of immune cells could be ability to stimulate otherimmune cells, killing activity, tumor-growth inhibitory activity, aswell as suppression of angiogenesis. Other means of assessingsuppression of immunity includes quantification of immune suppressivecells. For example, elevations in immature dendritic cells, Th2 cells,Th3 cells, myeloid suppressor cells, M2 macrophages, T regulatory cells,N2 neutrophils, and infiltration by mesenchymal stem cells possessingimmune suppressive properties, are all measurements for selectingpatients with immune suppression.

In some embodiments, extracorporeal removal of blocking factors isutilized to reduce the immune modulatory activity of myeloid suppressorcells as a means of inducing immunological activation. Myeloidsuppressor cells are believed to be similar to the “natural suppressor”cells described by the Singhal group in the 1970s. Natural suppressorcells were found to be bone marrow derived cells possessing ability toantigen-nonspecifically suppress T cell proliferation after immuneactivation [49-55], and are upregulated by cancer and pregnancy [56-63].These properties are similar to the currently described properties ofmyeloid derived suppressor cells [64].

In some embodiments of the invention, vitamin D3 is added toextracorporeal removal of blocking factors in order to augmentdifferentiation and/or loss of immune suppressive ability of saidmyeloid derived suppressor cells. Utilization of vitamin D3 to reducecancer associated immune suppression is described in this publicationand incorporated by reference [65, 66].

The invention teaches that in patients with pre-existing immunesuppression, removal of extracorporeal blocking factors may be used toincrease efficacy of checkpoint inhibitor drugs. For the practice of theinvention, various checkpoint inhibitors may be utilized together withextracorporeal removal of immunological blocking factors for enhancedtherapeutic activity. Examples of such checkpoint inhibitors include: a)Inhibitors of Programmed Death 1 (PD-1, CD279), such as nivolumab(OPDIVO™, BMS-936558, MDX1106, or MK-34775), and pembrolizumab(KEYTRUDA™, MK-3475, SCH-900475, lambrolizumab, CAS Reg. No.1374853-91-4), as well as the PD-1 blocking agents described in U.S.Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, WO03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO2011159877, WO 2011082400, and WO 2011161699; b) Inhibitors ofProgrammed Death-Ligand 1 (PD-L1, also known as B7-H1 and CD274),including antibodies such as BMS-936559, MPDL3280A), MEDI4736,MSB0010718C, and MDX1105-01); also including: atezolizumab, durvalumaband avelumab; c) Inhibitors of CTLA-4, such as ipilimumab (YERVOY™,MDX-010, BMS-734016, and MDX-101), tremelimumab, antibody clone BNI3(Abcam), RNA inhibitors, including those described in WO 1999/032619, WO2001/029058, U.S. 2003/0051263, U.S. 2003/0055020, U.S.2003/0056235,U.S. 2004/265839, U.S. 2005/0100913, U.S. 2006/0024798, U.S.2008/0050342, U.S. 2008/0081373, U.S. 2008/0248576, U.S. 2008/055443,U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095 and 7,560,438 (eachincorporated herein by reference); d) Inhibitors of PD-L2 (B7-DC,CD273), such as AMP-224 (Amplimune, Inc.) and rHIgM12B7; and e)Inhibitors of checkpoint proteins, including: LAG3, such as IMP321; TIM3(HAVCR2); 2B4; A2aR, ID02; B7H1; B7-H3 or B7H3, such as antibody MGA271;B7H4; BTLA; CD2; CD20, such as ibritumomab tiuxetan, ofatumumab,rituximab, obinutuzumab and tositumomab; CD27, such as CDX-1127; CD28;CD30, such as brentuximab vedotin; CD33, such as gemtuzumab ozogamicin;CD40; CD52, such as alemtuzumab; CD70; CD80; CD86; CD112; CD137; CD160;CD226; CD276; DR3; OX-40 (TNFRSF.sub.4 and CD134); GAL9; GITR; such asTRX518; HAVCR2; HVEM; IDI1; ICOS (inducible T cell costimulator; CD278);such as MEDI570 (MedImmune LLC) and AMG557 (Amgen); KR; LAIR; LIGHT;MARCO (macrophage receptor with collageneous structure); PS(phosphatidylserine); SLAM; TIGIT; VISTA; and VTCN1; or a combinationsthereof. In another variation, the checkpoint inhibitor is an inhibitorof a checkpoint protein selected from the group of PD-1, PD-L1, andCTLA-4. In another variation, the checkpoint inhibitor is selected fromthe group of an anti-PD-1 antibody, and anti-PD-L1 antibody, and ananti-CTLA-4 antibody. In one variation, the anti-PD-1 antibody isselected from the group of nivolumab, pembrolizumab, and lambrolizumab.In another variation, the anti-PD-L1 antibody is selected from the groupof as BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, and MDX1105-01. Inyet other variations, the anti-PD-L1 antibody is selected from the groupof durvalumab, atezolizumab, and avelumab. In another variation, theanti-CTLA-4 antibody is selected from the group of ipilimumab andtremelimumab. In one embodiment, the check point inhibitor is selectedfrom the group consisting of nivolumab, pembrolizumab, lambrolizumab,BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, durvalumab,atezolizumab, avelumab, ipilimumab, and tremelimumab. In certainembodiment, the check point inhibitor is selected from the groupconsisting of nivolumab, pembrolizumab, lambrolizumab, durvalumab,atezolizumab, avelumab, ipilimumab, and tremelimumab. In one embodiment,the check point inhibitor is selected from the group consisting ofnivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab. Saidcheckpoint inhibitors listed may be administered via multiple methods,including but not limited intravenously, intramuscularly, parenterally,nasally, intratumorally, intraosseously, subcutaneously, sublingually,intrarectally, intrathecally, intraventricularly, orally, intra-ocular,topically, or via inhalation, nanocell and/or nanobubble injection. Forpractices of the invention, the enhancement of efficacy of an immunecheckpoint inhibitor may be accomplished by performing one or moreclinical procedures involving the removal of tumor derived blockingfactors to prepare and/or condition the patient. Said removal may beperformed at various time points prior to administration of saidcheckpoint inhibitor(s). The determination of time points of removal, insome embodiments, is performed based on immunological and/or oncologicalassessment of the patient. In some situations, immune activity of thepatient is assessed, and used as a basis for determining the amount andfrequency of extracorporeal treatments prior to administration ofcheckpoint inhibitors. In some situations, it may be desirable tocontinue extracorporeal treatments while administering checkpointinhibitors. Furthermore, in some situations it may be desirable tocontinue extracorporeal treatment following administration of checkpointinhibitors.

In some embodiments, checkpoint inhibitor drugs are increased inefficacy by removal of extracorporeal blocking factors. Said checkpointinhibitor may be used to further increase in efficacy by addition of oneor more cancer vaccines, which is referred to as “active immunization”.In some embodiments of the invention, administration of checkpointinhibitors is performed together with active immunization. Immunizationmay take the form of peptides, proteins, altered peptide ligands, andcell therapy.

Antigens known to be found on cancer, and useful for the practice of theinvention include: epidermal growth factor receptor (EGFR, EGFR1,ErbB-1, HER1); ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligandfamily; insulin-like growth factor receptor (IGFR) family, IGF-bindingproteins (IGFBPs), IGFR ligand family (IGF-1R); platelet derived growthfactor receptor (PDGFR) family, PDGFR ligand family; fibroblast growthfactor receptor (FGFR) family, FGFR ligand family, vascular endothelialgrowth factor receptor (VEGFR) family, VEGF family; HGF receptor family;TRK receptor family; ephrin (EPH) receptor family; AXL receptor family;leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family,angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR)receptor family; discoidin domain receptor (DDR) family; RET receptorfamily; KLG receptor family; RYK receptor family; MuSK receptor family;transforming growth factor alpha (TGF-alpha), TGF-alphareceptor;transforming growth factor-beta (TGF-beta), TGF-beta receptor;interleukin beta receptor alpha2chain (IL13Ralpha2); interleukin-6(IL-6), IL-6 receptor; interleukin-4, IL-4 receptor; cytokine receptors,Class I (hematopoietin family) and Class II (interferon/IL-10 family)receptors; tumor necrosis factor (TNF) family, TNF-alpha; tumor necrosisfactor (TNF) receptor superfamily (TNTRSF); death receptor family,TRAIL-receptor; cancer-testis (CT) antigens; lineage-specific antigens;differentiation antigens; alpha-actinin-4; ARTC1, breakpoint clusterregion-Abelson (Bcr-abl) fusion products; B-RAF; caspasc-5 (CASP-5);caspase-8 (CASP-8); beta-catenin (CTNNB1); cell division cycle 27(CDC27); cyclin-dependent kinase 4 (CDK4); CDKN2A; COA-1; dek-can fusionprotein; EFTUD-2; Elongation factor 2 (ELF2); Ets variant gene 6/acutemyeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein; fibronectin(FN); GPNMB; low density lipid receptor/GDP-L fucose; beta-D-galactose2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein; HLA-A2; MLA-A11;heat shock protein 70-2 mutated (HSP70-2M); KIAA0205; MART2; melanomaubiquitous mutated 1, 2, 3 (MUM-1, 2, 3); prostatic acid phosphatase(PAP); neo-PAP; Myosin class 1; NFYC; OGT, OS-9; pml-RARalpha fusionprotein; PRDX5; PTPRK, K-ras (KRAS2); N-ras (NRAS); HRAS; RBAF600;SIRT12; SNRPD1; SYT-SSX1 or -SSX2 fusion protein; TriosephosphateIsomerase; BAGE; BAGE-1; BAGE-2, 3, 4, 5; GAGE-1, 2, 3, 4, 5, 6, 7, 8;GnT-V (aberrant N-acetyl glucosaminyl transferase V; MGAT5), HERV-K MEL,KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma(CAMEL), MAGE-A1 (MAGE-1); MAGE-A2; MAGE-A3; MAGE-A4; MAGE-AS; MAGE-A6;MAGE-A8; MAGE-A9; MAGE-A10; MAGE-A11; MAGE-A12; MAGE-3; MAGE-B1;MAGE-B2; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; mucin 1 (MUC1);MART-1/Melan-A (MLANA); gp100; gp100/Pme117 (S1LV); tyrosinase (TYR);TRP-1; HAGE; NA-88; NY-ESO-1; NY-ESO-1/LAGE-2; SAGE, Sp17; SSX-1, 2, 3,4; TRP2-1NT2; carcino-embryonic antigen (CEA); Kallikrein 4;mammaglobin-A; OA1; prostate specific antigen (PSA); prostate specificmembrane antigen; TRP-1/, 75; TRP-2 adipophilin; interferon inducibleprotein absent in melanoma 2 (AIM-2); BING-4; CPSF; cyclin D1;epithelial cell adhesion molecule (Ep-CAM); EpbA3; fibroblast growthfactor-5 (FGF-5); glycoprotein 250 (gp250intestinal carboxyl esterase(iCE); alpha-feto protein (AFP); M-CSF; mdm-2; MUCI; p53 (TP53); PBF;PRAME; PSMA; RAGE-1; RNF43; RU2AS; SOX10; STEAP1; survivin (MRCS); humantelomerase reverse transcriptase (hTERT); telomerase; Wilms' tumor gene(WT1); SYCP1; BRDT; SPANX; XAGE; ADAM2; PAGE-5; LIP1; CTAGE-1; CSAGE;MMA1; CAGE; BORIS; HOM-TES-85; AF15q14; HCA661; LDHC; MORC; SGY-1;SPO11; TPX1; NY-SAR-35; FTHLI7; NXF2TDRD1; TEX 15; FATE; TPTE;immunoglobulin idiotypes; Bence-Jones protein; estrogen receptors (ER);androgen receptors (AR); CD40; CD30; CD20; CD19; CD33; CD4; CD25; CD3;cancer antigen 72-4 (CA 72-4); cancer antigen 15-3 (CA 15-3); cancerantigen 27-29 (CA 27-29); cancer antigen 125 (CA 125); cancer antigen19-9 (CA 19-9); beta-human chorionic gonadotropin; 1-2 microglobulin;squamous cell carcinoma antigen; neuron-specific enolase; heat shockprotein gp96; GM2, sargramostim; CTLA-4; 707 alanine proline (707-AP);adenocarcinoma antigen recognized by T cells 4 (ART-4);carcinoembryogenic antigen peptide-1 (CAP-1); calcium-activated chloridechannel-2 (CLCA2); cyclophilin B (Cyp-B); and human signet ring tumor-2(HST-2).

In one embodiment, the invention teaches the use of removal ofextracorporeal blocking factors to increase the number of dendriticcells infiltrating tumors. The utilization of dendritic cells as animmunotherapy is known in the art and ways of using dendritic celltherapy are defined in the following examples for melanoma [67-118],soft tissue sarcoma [119], thyroid [120-122], glioma [123-144], multiplemyeloma ,[145-153], lymphoma [154-156], leukemia [157-164], as well asliver [165-170], lung [171-184], ovarian [185-188], and pancreaticcancer [189-191]. In other embodiments the invention teaches the use ofextracorporeal removal of immunological blocking factors foraugmentation of existing dendritic cells to infiltrate tumors. Means ofassessing dendritic cell infiltration are known in the art and describedin the following examples: for gastric cancer [192-195], head and neckcancer [196-200], cervical cancer [201], breast cancer [202-204], lungcancer [205], colorectal cancer [206-208], liver cancer [209, 210], gallbladder cancer [211, 212], and pancreatic cancer [213].

REFERENCES

-   1. Mukherjee, N., K. M . Wheeler, and R. S. Svatek, Bacillus    Calmette-Guerin treatment of bladder cancer: a systematic review and    commentary on recent publications. Curr Opin Urol, 2019.-   2. Lemdani, K., et al., Local immunomodulation combined to    radiofrequency ablation results in a complete cure of local and    distant colorectal carcinoma. Oncoimmunology, 2019. 8(3): p.    1550342.-   3. Pettenati, C. and M. A. Ingersoll, Mechanisms of BCG    immunotherapy and its outlook for bladder cancer. Nat Rev    Urol, 2018. 15(10): p. 615-625.-   4. Mukherjee, N. and R. Svatek, Cancer Immune Therapy: Prognostic    Significance and Implications for Therapy of PD-1 in BCG-Relapsing    Bladder Cancer. Ann Surg Oncol, 2018. 25(9): p. 2498-2499.-   5. Godoy-Calderon, M. J., et al., Autologous tumor cells/bacillus    Calmette-Guerin/formalin-based novel breast cancer vaccine induces    an inzmune antitumor response. Oncotarget, 2018. 9(29): p.    20222-20238.-   6. Davis, R. L., 3rd, W. Le, and Z. Cui, Granulocytes as an effector    mechanism of BCG therapy for bladder cancer. Med Hypotheses, 2017.    104: p. 166-169.-   7. Morales, A., BCG: A throwback from the stone age of vaccines    opened the path for bladder cancer immunotherapy. Can J Urol, 2017.    24(3): p. 8788-8793.-   8. Kowalewicz-Kulbat, M. and C. Locht, BCG and protection against    inflammatory and auto-immune diseases. Expert Rev Vaccines, 2017.    16(7): p. 1-10.-   9. Prack Mc Cormick, B., et al., Bacillus Calmette-Guerin improves    local and systemic response to radiotherapy in invasive bladder    cancer. Nitric Oxide, 2017. 64: p. 22-30.-   10. Pichler, R., et al., Tumor-infiltrating immune cell    subpopulations influence the oncologic outcome after intravesical    Bacillus Calmette-Guerin therapy in bladder cancer.    Oncotarget, 2016. 7(26): p. 39916-39930.-   11. Pan, K., et al., OK-432 synergizes with IFN-gamma to confer    dendritic cells with enhanced antitumor immunity. Immunol Cell    Biol, 2014. 92(3): p. 263-74.-   12. Ohe, G., et al., Effect of soluble factors derived from oral    cancer cells on the production of interferon-gamma from peripheral    blood mononuclear cells following stimulation with OK-432. Oncol    Rep, 2013. 30(2): p. 945-51.-   13. Hirayama, M., et al., Overcoming regulatory T-cell suppression    by a lyophilized preparation of Streptococcus pyogenes. Eur J    Immunol, 2013. 43(4): p. 989-1000.-   14. Chen, U., et al., Vaccination with OK-432 followed by TC-1 tumor    lysate leads to significant antitumor effects. Reprod Sci, 2011.    18(7): p. 687-94.-   15. Oshikawa, T., et al., Antitumor effect of OK-432-derived DNA:    one of the active constituents of OK-432, a streptococcal    immunotherapeutic agent. J Immunother, 2006. 29(2): p. 143-50.-   16. Okamoto, M., et al., Mechanism of anticancer host response    induced by OK-432, a streptococcal preparation, mediated by    phagocytosis and Toll-like receptor 4 signaling. J Immunother, 2006.    29(1): p. 78-86.-   17. Kimura, T., et al., Final report of a randomized controlled    study with streptococcal preparation OK-432 as a supplementary    immunopotentiator for laryngeal cancer. Acta Otolaryngol    Suppl, 1996. 525: p. 135-41.-   18. Ozaki, S., et al., Mechanism of tumoricidal activity of    OK-432-specific L3T4+Lyt2-T-cells. Cancer Res, 1990. 50(15): p.    4630-4.-   19. Abe, Y., et al., The endogenous induction of tumor necrosis    factor serum (TNS) for the adjuvant postoperative immunotherapy of    cancer—changes in immunological markers of the blood. Jpn J    Surg, 1990. 20(1): p. 19-26.-   20. Nio, Y., et al., Cytotoxic and cytostatic effects of the    streptococcal preparation OK-432 and its subcellular fractions on    human ovarian tumor cells. Cancer, 1989. 64(2): p. 434-41.-   21. Hanaue, H., et al., Hemolytic streptococcus preparation OK-432;    beneficial adjuvant therapy in recurrent gastric carcinoma. Tokai J    Exp Clin Med, 1987. 12(4): p. 209-14.-   22. Ujiie, T., OK-432-mediated augmentation of antitumor immunity    and generation of cytotoxic T lymphocytes. Jpn J Exp Med, 1987.    57(2): p. 103-15.-   23. Bonavida, B., J. Katz, and T. Hoshino, Mechanism of NK    activation by OK-432 (Streptococcus pyogenes). I. Spontaneous    release of NKCF and augmentation of NKCF production following    stimulation with NK target cells. Cell Immunol, 1986. 102(1): p.    126-35.-   24. Uchida, A., Augmentation of autologous tumor killing activity of    tumor-associated large granular lymphocytes by the streptococcal    preparation OK432. Methods Find Exp Clin Pharmacol, 1986. 8(2): p.    81-4.-   25. Toge, T., et al., Effects of intraperitoneal administration of    OK-432 for patients with advanced cancer. Jpn J Surg, 1985.    15(4): p. 260-5.-   26. Sakai, S., et al., Studies on the properties of a streptococcal    preparation OK-432 (NSC-B116209) as an immunopotentiator. I.    Activation of serum complement components and peritoneal exudate    cells by group A streptococcus. Jpn J Exp Med, 1976. 46(2): p.    123-33.-   27. Pampena, M. B., et al., Dissecting the Immune Stimulation    Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma    Patients: Long Term Antitumor Immunity and Short Term Release of    Acute Inflanzmatoty Reactants. Front Immunol, 2018. 9: p. 2531.-   28. Pampena, M. B., et al., Early Events of the Reaction Elicited by    CSF-470 Melanoma Vaccine Plus Adjuvants: An In Vitro Analysis of    Immune Recruitment and Cytokine Release. Front Immunol, 2017. 8: p.    1342.-   29. Lutsiak, M. E., et al., Inhibition of CD4(+)25+ T regulatory    cell function implicated in enhanced immune response by low-dose    cyclophosphamide. Blood, 2005. 105(7): p. 2862-8.-   30. Beyer, M., et al., Reduced frequencies and suppressive function    of CD4+CD25hi regulatory T cells in patients with chronic    lymphocytic leukemia after therapy with fludarabine. Blood, 2005.    106(6): p. 2018-25.-   31. Brode, S., et al., Cyclophosphamide-induced type-I diabetes in    the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+    regulatory T cells. J Immunol, 2006. 177(10): p. 6603-12.-   32. Salem, M. L., et al., Defining the ability of cyclophosphamide    preconditioning to enhance the antigen-specific CD8+ T-cell response    to peptide vaccination: creation of a beneficial host    microenvironment involving type I IFNs and myeloid cells. J    Immunother, 2007. 30(1): p. 40-53.-   33. Liu, J .Y., et al., Single administration of low dose    cyclophosphamide augments the antitumor effect of dendritic cell    vaccine. Cancer Immunol Immunother, 2007. 56(10): p. 1597-604.-   34. van der Most, R. G., et al., Tumor eradication after    cyclophosphamide depends on concurrent depletion of regulatory T    cells: a role for cycling TN FR2-expressing effector-suppressor T    cells in limiting effective chemotherapy. Cancer Immunol    Immunother, 2009. 58(8): p. 1219-28.-   35. Greten, T. F., et al., Low-dose cyclophosphamide treatment    impairs regulatory T cells and unmasks AFP-specific CD4+ T-cell    responses in patients with advanced HCC. J Immunother, 2010.    33(2): p. 211-8.-   36. Zhao, J., et al., Selective depletion of CD4+CD25+Foxp3+    regulatory T cells by low-dose cyclophosphamide is explained by    reduced intracellular ATP levels. Cancer Res, 2010. 70(12): p.    4850-8.-   37. Vermeij, R., et al., Potentiation of a p53-SLP vaccine by    cyclophosphamide in ovarian cancer: a single arm phase II study. Int    J Cancer, 2012. 131(5): p. E670-80.-   38. Xia, Q., et al., Cyclophosphamide enhances anti-tumor effects of    a fibroblast activation protein alpha-based DNA vaccine in    tumor-bearing mice with murine breast carcinoma. Immunopharmacol    Immunotoxicol, 2017. 39(1): p. 37-44.-   39. Noordam, L., et al., Low-dose cyclophosphamide depletes    circulating naive and activated regulatory T cells in malignant    pleural mesothelioma patients synergistically treated with dendritic    cell-based immunotherapy. Oncoimmunology, 2018. 7(12): p. e1474318.-   40. Lieberman, R., J. Wybran, and W. Epstein, The immunologic and    histopathologic changes of BCG-mediated tumor regression in patients    with malignant melanoma. Cancer, 1975. 35(3): p. 756-77.-   41. Leach, D. R., M. F. Krurnmel, and J. P. Allison, Enhancement of    antitumor immunity by CTLA-4 blockade. Science, 1996. 271(5256): p.    1734-6.-   42. Zhang, D., et al., Scoring System for Tumor-Infiltrating    Lymphocytes and Its Prognostic Value for Gastric Cancer. Front    Immunol, 2019. 10: p. 71.-   43. Huang, J., et al., Changes of Tumor Infiltrating Lymphocytes    after Core Needle Biopsy and the Prognostic Implications in Early    Stage Breast Cancer: A Retrospective Study. Cancer Res Treat, 2019.-   44. Miyoshi, Y., et al., Associations in tumor infiltrating    lymphocytes between clinicopathological factors and clinical    outcomes in estrogen receptor-positive/human epidermal growth factor    receptor type 2 negative breast cancer. Oncol Lett, 2019. 17(2): p.    2177-2186.-   45. Loi, S., et al., Tumor-Infiltrating Lymphocytes and Prognosis: A    Pooled Individual Patient Analysis of Early-Stage Triple Negative    Breast Cancers. J Clin Oncol, 2019. 37(7): p. 559-569.-   46. Sonderstrup, I. M. H., et al., Evaluation of tumor-infiltrating    lymphocytes and association with prognosis in BRCA-mutated breast    cancer. Acta Oncol, 2019: p. 1-8.-   47. Wong, P .F., et al., Multiplex Quantitative Analysis of    Tumor-Infiltrating Lymphocytes and Immunotherapy Outcome in    Metastatic Melanoma. Clin Cancer Res, 2019.-   48. Shimizu, S., et al., Tumor-infiltrating CD8(+) T-cell density is    an independent prognostic marker for oral squamous cell carcinoma.    Cancer Med, 2019. 8(1): p. 80-93.-   49. Bennett, J. A., V. S. Rao, and M. S. Mitchell, Systemic bacillus    Calmette-Guerin (BCG) activates natural suppressor cells. Proc Natl    Acad Sci U S A, 1978. 75(10): p. 5142-4.-   50. Bennett, J. A. and J. C. Marsh, Relationship of Bacillus    Calmette-Guerin-induced suppressor cells to hematopoietic precursor    cells. Cancer Res, 1980. 40(1): p. 80-5.-   51. Strober, S., Natural suppressor (NS) cells, neonatal tolerance,    and total lymphoid irradiation: exploring obscure relationships.    Annu Rev Immunol, 1984. 2: p. 219-37.-   52. Schwadron, R. B., D. M. Gandour, and S. Strober, Cloned natural    suppressor cell lines derived from the spleens of neonatal mice. J    Exp Med, 1985. 162(1): p. 297-310.-   53. Noga, S. J., et al., Characterization of the natural suppressor    cell population in adult rat bone marrow. J Leukoc Biol, 1988.    43(3): p. 279-87.-   54. Sugiura, K., et al., Enrichment of natural suppressor activity    in a wheat germ agglutinin positive hematopoietic    progenitor-enriched fraction of monkey bone marrow. Blood, 1990.    75(5): p. 1125-31.-   55. Sugiura, K., et al., Enrichment of murine bone marrow natural    suppressor activity in the fraction of hematopoietic progenitors    with interleukin 3 receptor-associated antigen. Exp Hematol, 1992.    20(2): p. 256-63.-   56. Clark, D. A., et al., Decidua-associated suppressor cells in    abortion-prone DBA/2-mated CBA/J mice that release bioactive    transforming growth factor beta2-related immunosuppressive molecules    express a bone marrow-derived natural suppressor cell marker and    gamma delta T-cell receptor. Biol Reprod, 1997. 56(5): p. 1351-60.-   57. Gronvik, K. O., D. W. Hoskin, and R. A. Murgita, Monoclonal    antibodies against murine neonatal and pregnancy-associated natural    suppressor cells induce resorption of the fetus. Scand J    Immunol, 1987. 25(5): p. 533-40.-   58. Subiza, J. L., et al., Development of splenic natural suppressor    (NS) cells in Ehrlich tumor-bearing mice. Int J Cancer, 1989.    44(2): p. 307-14.-   59. Vinuela, J. E., et al., Antigen shedding vs. development of    natural suppressor cells as mechanism of tumor escape in mice    bearing Ehrlich tumor. Int J Cancer, 1991. 47(1): p. 86-91.-   60. Hayamizu, K., et al., Induction of natural suppressor-like cells    from human adult peripheral blood lymphocytes by a K562-derived    factor. Transplantation, 1993. 55(6): p. 1403-8.-   61. Prechel, M. M., et al., Immune modulation by interleukin-12 in    tumor-bearing mice receiving vitamin D3 treatments to block    induction of immunosuppressive granulocyte/macrophage progenitor    cells. Cancer Immunol Immunother, 1996. 42(4): p. 213-20.-   62. Young, M. R., et al., Increased recurrence and metastasis in    patients whose prinzary head and neck squamous cell carcinomas    secreted granulocyte-macrophage colony-stimulating factor and    contained CD34+ natural suppressor cells. Int J Cancer, 1997.    74(1): p. 69-74.-   63right, M. ., et al., mulation of immune suppressive CD34+ cells    from normal bone marrow by Lewis lung carcinoma tumors. Cancer    Immunol Immunother, 1998. 46(5): p. 253-60.-   64. Martino, A., et al., Mycobacterium bovis bacillus    Calmette-Guerin vaccination mobilizes innate myeloid-derived    suppressor cells restraining in vivo T cell priming via    IL-1R-dependent nitric oxide production. J Immunol, 2010. 184(4): p.    2038-47.-   65. Wiers, K., et al., Failure of tumor-reactive lymph node cells to    kill tumor in the presence of immune-suppressive CD34+ cells can be    overcome with vitamin 3 treatment to diminish CD34+ cell levels.    Clin Exp Metastasis, 1998. 16(3): p. 275-82.-   66. Wiers, K. M., et al., Vitamin D3 treatment to diminish the    levels of immune suppressive CD34+ cells increases the effectiveness    of adoptive immunotherapy. J Immunother, 2000. 23(1): p. 115-24.-   67. Nestle, F. O., et al., Vaccination of melanoma patients with    peptide-or tumor lysate-pulsed dendritic cells. Nat Med, 1998.    4(3): p. 328-32.-   68. Chakraborty, N. G., et al., Immunization with a    tumor-cell-lysate-loaded autologous-antigen-presenting-cell-based    vaccine in melanoma. Cancer Immunol Immunother, 1998. 47(1): p.    58-64.-   69. Wang, F., et al., Phase I trial of a MART-1 peptide vaccine with    incomplete Freund's adjuvant for resected high-risk melanoma. Clin    Cancer Res, 1999. 5(10): p. 2756-65.-   70. Thurner, B., et al., Vaccination with mage-3A1 peptide-pulsed    mature, monocyte-derived dendritic cells expands specific cytotoxic    T cells and induces regression of some metastases in advanced stage    IV melanoma. J Exp Med, 1999. 190(11): p. 1669-78.-   71. Thomas, R., et al., Immature human monocyte-derived dendritic    cells migrate rapidly to draining lymph nodes after intradermal    injection for melanoma immunotherapy. Melanoma Res, 1999. 9(5): p.    474-81.-   72. Mackensen, A., et al., Phase I study in melanoma patients of a    vaccine with peptide-pulsed dendritic cells generated in vitro from    CD34(+) hematopoietic progenitor cells. Int J Cancer, 2000.    86(3): p. 385-92.-   73. Panelli, M. C., et al., Phase I study in patients with    metastatic melanoma of immunization with dendritic cells presenting    epitopes derived from the melanoma-associated antigens MART-1 and    gp100. J Immunother, 2000. 23(4): p. 487-98.-   74. Schuler-Thurner, B., et al., Mage-3 and influenza-matrix    peptide-specific cytotoxic T cells are inducible in terminal stage    HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic    cells. J Immunol, 2000. 165(6): p. 3492-6.-   75. Lau, R., et al., Phase I trial of intravenous peptide-pulsed    dendritic cells in patients with metastatic melanoma. J    Immunother, 2001. 24(1): p. 66-78.-   76. Banchereau, J., et al., Immune and clinical responses in    patients with metastatic melanoma to CD34(+) progenitor-derived    dendritic cell vaccine. Cancer Res, 2001. 61(17): p.6451-8.-   77. Schuler-Thurner, B., et al., Rapid induction of tumor-specific    type 1 T helper cells in metastatic melanoma patients by vaccination    with mature, cryopreserved, peptide-loaded monocyte-derived    dendritic cells. J Exp Med, 2002. 195(10): p. 1279-88.-   78. Palucka, A. K., et al., Single injection of CD34+    progenitor-derived dendritic cell vaccine can lead to induction of    T-cell immunity in patients with stage IV melanoma. J    Immunother, 2003. 26(5): p. 432-9.-   79. Bedrosian, I., et al., Intranodal administration of    peptide-pulsed mature dendritic cell vaccines results in superior    CD8+ T-cell function in melanoma patients. J Clin Oncol, 2003.    21(20): p. 3826-35.-   80. Slingluff, C. L., Jr., et al., Clinical and immunologic results    of a randomized phase II trial of vaccination using four melanoma    peptides either administered in granulocyte-macrophage    colony-stimulating factor in adjuvant or pulsed on dendritic cells.    J Clin Oncol, 2003. 21(21): p. 4016-26.-   81. Hersey, P., et al., Phase I/II study of treatment with dendritic    cell vaccines in patients with disseminated melanoma. Cancer Immunol    Immunother, 2004. 53(2): p. 125-34.-   82. Vilella, R., et al., Pilot study of treatment of    biochemotherapy-refractory stage IV melanoma patients with    autologous dendritic cells pulsed with a heterologous melanoma cell    line lysate. Cancer Immunol Immunother, 2004. 53(7): p. 651-8.-   83. Palucka, A. K., et al., Spontaneous proliferation and type 2    cytokine secretion by CD4+ T cells in patients with metastatic    melanoma vaccinated with antigen-pulsed dendritic cells. J Clin    Immunol, 2005. 25(3): p. 288-95.-   84. Banchereau, J., et al., Immune and clinical outcomes in patients    with stage IV melanoma vaccinated with peptide-pulsed dendritic    cells derived from CD34+ progenitors and activated with type I    interferon. J Immunother, 2005. 28(5): p. 505-16.-   85. Trakatelli, M., et al., A new dendritic cell vaccine generated    with interleukin-3 and interferon-beta induces CD8+ T cell responses    against NA 17-A2 tumor peptide in melanoma patients. Cancer Immunol    Immunother, 2006. 55(4): p. 469-74.-   86. Salcedo, M., et al., Vaccination of melanoma patients using    dendritic cells loaded with an allogeneic tumor cell lysate. Cancer    Immunol Immunother, 2006. 55(7): p. 819-29.-   87. Linette, G. P., et al., Immunization using autologous dendritic    cells pulsed with the melanoma-associated antigen gp100-derived    G280-9V peptide elicits CD8+ immunity. Clin Cancer Res, 2005.    11(21): p. 7692-9.-   88. Escobar, A., et al., Dendritic cell immunizations alone or    combined with low doses of interleukin-2induce specific inzinune    responses in melanoma patients. Clin Exp Immunol, 2005. 142(3): p.    555-68.-   89. Tuettenberg, A., et al., Induction of strong and persistent    MelanA/MART-1-specific immune responses by adjuvant dendritic    cell-based vaccination of stage II melanoma patients. Int J    Cancer, 2006. 118(10): p. 2617-27.-   90. Schadendorf, D., et al., Dacarbazine (DTIC) versus vaccination    with autologous peptide-pulsed dendritic cells (DC)in first-line    treatment of patients with metastatic melanoma: a randomized phase    III trial of the DC study group of the DeCOG. Ann Oncol, 2006.    17(4): p. 563-70.-   91. Di Pucchio, T., et al., Immunization of stage IV melanoma    patients with Melan-A/MART-1 and gp100 peptides plus IFN-alpha    results in the activation of specific CD8(+) T cells and    monocyte/dendritic cell precursors. Cancer Res, 2006. 66(9): p.    4943-51.-   92. Nakai, N., et al., Vaccination of Japanese patients with    advanced melanoma with peptide, tumor lysate or both peptide and    tumor lysate-pulsed mature, monocyte-derived dendritic cells. J    Dermatol, 2006. 33(7): p. 462-72.-   93. Palucka, A. K., et al., Dendritic cells loaded with killed    allogeneic melanoma cells can induce objective clinical responses    and MART-1 specific CD8+ T-cell immunity. J Immunother, 2006.    29(5): p. 545-57.-   94. Lesimple, T., et al., Immunologic and clinical effects of    injecting mature peptide-loaded dendritic cells by intralymphatic    and intranodal routes in metastatic melanoma patients. Clin Cancer    Res, 2006. 12(24): p. 73800-8.-   95. Guo, J., et al., Intratumoral injection of dendritic cells in    combination with local hyperthermia induces systemic antitumor    effect in patients with advanced melanoma. Int J Cancer, 2007.    120(11): p. 2418-25.-   96. O'Rourke, M. G., et al., Dendritic cell immunotherapy for stage    IV melanoma. Melanoma Res, 2007. 17(5): p. 316-22.-   97. Bercovici, N., et al., Analysis and characterization of    antitumor T-cell response after administration of dendritic cells    loaded with allogeneic tumor lysate to metastatic melanoma patients.    J Immunother, 2008. 31(1): p. 101-12.-   98. Hersey, P., et al., Phase I/II study of treatment with matured    dendritic cells with or without low dose IL-2 in patients with    disseminated melanoma. Cancer Immunol Immunother, 2008. 57(7): p.    1039-51.-   99. von Euw, E. M., et al., A phase I clinical study of vaccination    of melanoma patients with dendritic cells loaded with allogeneic    apoptotic/necrotic melanoma cells. Analysis of toxicity and immune    response to the vaccine and of IL-10-1082 promoter genotype as    predictor of disease progression. J Transl Med, 2008. 6: p. 6.-   100. Carrasco, J., et al., Vaccination of a melanoma patient with    mature dendritic cells pulsed with MAGE-3 peptides triggers the    activity of nonvaccine anti-tumor cells. J Immunol, 2008. 180(5): p.    3585-93.-   101. Redman, B. G., et al., Phase Ib trial assessing autologous,    tumor-pulsed dendritic cells as a vaccine administered with or    without IL-2 in patients with metastatic melanoma. J    Immunother, 2008. 31(6): p. 591-8.-   102. Daud, A. I., et al., Phenotypic and functional analysis of    dendritic cells and clinical outcome in patients with high-risk    melanoma treated with adjuvant granulocyte macrophage    colony-stimulating factor. J Clin Oncol, 2008. 26(19): p. 3235-41.-   103. Engell-Noerregaard, L., et al., Review of clinical studies on    dendritic cell-based vaccination of patients with malignant    melanoma: assessment of correlation between clinical response and    vaccine parameters. Cancer Immunol Immunother, 2009. 58(1): p. 1-14.-   104. Nakai, N., et al., Immunohistological analysis of    peptide-induced delayed-type hypersensitivity in advanced melanoma    patients treated with melanoma antigen-pulsed mature    monocyte-derived dendritic cell vaccination. J Dermatol Sci, 2009.    53(1): p. 40-7.-   105. Dillman, R. O., et al., Phase II trial of dendritic cells    loaded with antigens from self renewing, proliferating autologous    tumor cells as patient-specific antitumor vaccines in patients with    metastatic melanoma: final report. Cancer Biother Radiophann, 2009.    24(3): p. 311-9.-   106. Chang, J. W., et al., Immunotherapy with dendritic cells pulsed    by autologous dactinomycin-induced melanoma apoptotic bodies for    patients with malignant melanoma. Melanoma Res, 2009. 19(5): p.    309-15.-   107. Trepiakas, R., et al., Vaccination with autologous dendritic    cells pulsed with multiple tumor antigens for treatment of patients    with malignant melanoma: results from a phase I/II trial.    Cytotherapy, 2010. 12(6): p. 721-34.-   108. Jacobs, J. F., et al., Dendritic cell vaccination in    combination with anti-CD25 monoclonal antibody treatment: a phase    I/II study in metastatic melanoma patients. Clin Cancer Res, 2010.    16(20): p. 5067-78.-   109. Ribas, A., et al., Multicenter phase II study of matured    dendritic cells pulsed with melanoma cell line lysates in patients    with advanced melanoma. J Transl Med, 2010. 8: p. 89.-   110. Ridolfi, L., et al., Unexpected high response rate to    traditional therapy after dendritic cell-based vaccine in advanced    melanoma: update of clinical outcome and subgroup analysis. Clin Dev    Immunol, 2010. 2010: p. 504979.-   111. Cornforth, A. N., et al., Resistance to the proapoptotic    effects of interferon-gamma on melanoma cells used in    patient-specific dendritic cell immunotherapy is associated with    improved overall survival. Cancer Immunol Immunother, 2011.    60(1): p. 123-31.-   112. Lesterhuis, W. J., et al., Wild-type and modified gp100    peptide-pulsed dendritic cell vaccination of advanced melanoma    patients can lead to long-term clinical responses independent of the    peptide used. Cancer Immunol Immunother, 2011. 60(2): p. 249-60.-   113. Bjoem, J., et al., Changes in peripheral blood level of    regulatory T cells in patients with malignant melanoma during    treatment with dendritic cell vaccination and low-dose IL-2. Scand J    Immunol, 2011. 73(3): p. 222-33.-   114. Steele, J. C., et al., Phase I/II trial of a dendritic cell    vaccine transfected with DNA encoding melan A and gp100 for patients    with metastatic melanoma. Gene Ther, 2011. 18(6): p. 584-93.-   115. Kim, D. S., et al., Immunotherapy of malignant melanoma with    tumor lysate-pulsed autologous monocyte-derived dendritic cells.    Yonsei Med J, 2011. 52(6): p. 990-8.-   116. Ellebaek, E., et al., Metastatic melanoma patients treated with    dendritic cell vaccination, Interleukin-2 and metronomic    cyclophosphamide: results from a phase II trial. Cancer Immunol    Immunother, 2012. 61(10): p. 1791-804.-   117. Dillman, R. O., et al., Tumor stem cell antigens as    consolidative active specific immunotherapy: a randomized phase II    trial of dendritic cells versus tumor cells in patients with    metastatic melanoma. J Immunother, 2012. 35(8): p. 641-9.-   118. Dannull, J., et al., Melanoma immunotherapy using mature DCs    expressing the constitutive proteasome. J Clin Invest, 2013.    123(7): p. 3135-45.-   119. Finkelstein, S. E., et al., Combination of external beam    radiotherapy (EBRT) with intratumoral injection of dendritic cells    as neo-adjuvant treatment of high-risk soft tissue sarcoma patients.    Int J Radial Oncol Biol Phys, 2012. 82(2): p. 924-32.-   120. Stift, A., et al., Dendritic cell vaccination in medullary    thyroid carcinoma. Clin Cancer Res, 2004. 10(9): p. 2944-53.-   121. Kuwabara, K., et al., Results of a phase I clinical study using    dendritic cell vaccinations for thyroid cancer. Thyroid, 2007.    17(1): p. 53-8.-   122. Bachleitner-Hofmann, T., et al., Pilot trial of autologous    dendritic cells loaded with tumor lysate(s) from allogeneic tumor    cell lines in patients with metastatic medullary thyroid carcinoma.    Oncol Rep, 2009. 21(6): p. 1585-92.-   123. Yu, J. S., et al., Vaccination of malignant glioma patients    with peptide-pulsed dendritic cells elicits systemic cytotoxicity    and intracranial T-cell infiltration. Cancer Res, 2001. 61(3): p.    842-7.-   124. Yamanaka, R., et al., Vaccination of recurrent glioma patients    with tumour lysate-pulsed dendritic cells elicits immune responses:    results of a clinical phase I/II trial. Br J Cancer, 2003. 89(7): p.    1172-9.-   125. Yu, J. S., et al., Vaccination with tumor lysate-pulsed    dendritic cells elicits antigen-specific, cytotoxic T-cells in    patients with malignant glioma. Cancer Res, 2004. 64(14): p. 4973-9.-   126. Yamanaka, R., et al., Tumor lysate and IL-18 loaded dendritic    cells elicits Th1 response, tumor-specific CD8+ cytotoxic T cells in    patients with malignant gnome. J Neurooncol, 2005. 72(2): p. 107-13.-   127. Yamanaka, R., et al., Clinical evaluation of dendritic cell    vaccination for patients with recurrent glioma: results of a    clinical phase I/II trial. Clin Cancer Res, 2005. 11(11): p. 4160-7.-   128. Liau, L. M., et al., Dendritic cell vaccination in glioblastoma    patients induces systemic and intracranial T-cell responses    modulated by the local central nervous system tumor    microenvironment. Clin Cancer Res, 2005. 11(15): p. 5515-25.-   129. Walker, D. G., et al., Results of a phase I dendritic cell    vaccine trial for malignant astrocytoma: potential interaction with    adjuvant chemotherapy. J Clin Neurosci, 2008. 15(2): p. 114-21.-   130. Leplina, O. Y., et al., Use of interferon-alpha-induced    dendritic cells in the therapy of patients with malignant brain    gliomas. Bull Exp Biol Med, 2007. 143(4): p. 528-34.-   131. De Vleeschouwer, S., et al., Postoperative adjuvant dendritic    cell-based immunotherapy in patients with relapsed glioblastoma    multiforme. Clin Cancer Res, 2008. 14(10): p. 3098-104.-   132. Ardon, H., et al., Adjuvant dendritic cell-based tumour    vaccination for children with malignant brain tumours. Pediatr Blood    Cancer, 2010. 54(4): p. 519-25.-   133. Prins, R. M., et al., Gene expression profile correlates with    T-cell infiltration and relative survival in glioblastoma patients    vaccinated with dendritic cell immunotherapy. Clin Cancer Res, 2011.    17(6): p. 1603-15.-   134. Okada, H., et al., Induction of CD8+ T-cell responses against    novel glioma-associated antigen peptides and clinical activity by    vaccinations with {alpha}-type 1 polarized dendritic cells and    polyinosinic-polycytidylic acid stabilized by lysine and    carboxymethylcellulose in patients with recurrent malignant glioma.    J Clin Oncol, 2011. 29(3): p. 330-6.-   135. Fadul, C. E., et al., Immune response in patients with newly    diagnosed glioblastoma multiforme treated with intranodal autologous    tumor lysate-dendritic cell vaccination after radiation    chemotherapy. J Immunother, 2011. 34(4): p. 382-9.-   136. Chang, C. N., et al., A phase I/II clinical trial investigating    the adverse and therapeutic effects of a postoperative autologous    dendritic cell tumor vaccine in patients with malignant glioma. J    Clin Neurosci, 2011. 18(8): p. 1048-54.-   137. Cho, D. Y., et al., Adjuvant immunotherapy with whole-cell    lysate dendritic cells vaccine for glioblastoma multiforme: a phase    II clinical trial. World Neurosurg, 2012. 77(5-6): p. 736-44.-   138. Iwami, K., et al., Peptide-pulsed dendritic cell vaccination    targeting interleukin-13 receptor alpha2 chain in recurrent    malignant glioma patients with HLA-A*24/A*02 allele.    Cytotherapy, 2012. 14(6): p. 733-42.-   139. Fong, B., et al., Monitoring of regulatory T cell frequencies    and expression of CTLA-4 on T cells, before and after DC    vaccination, can predict survival in GBM patients. PLoS One, 2012.    7(4): p. e32614.-   140. De Vleeschouwer, S., et al., Stratification according to    HGG-IMMUNO RPA model predicts outcome in a large group of patients    with relapsed malignant glioma treated by adjuvant postoperative    dendritic cell vaccination. Cancer Immunol Immunother, 2012.    61(11): p. 2105-12.-   141. Phuphanich, S., et al., Phase I trial of a multi-epitope-pulsed    dendritic cell vaccine for patients with newly diagnosed    glioblastoma. Cancer Immunol Immunother, 2013. 62(1): p. 125-35.-   142. Akiyama, Y., et al., alpha-type-1 polarized dendritic    cell-based vaccination in recurrent high-grade glioma: a phase I    clinical trial. BMC Cancer, 2012. 12: p. 623.-   143. Prins, R. M., et al., Comparison of glioma-associated antigen    peptide-loaded versus autologous tumor lysate-loaded dendritic cell    vaccination in malignant glioma patients. J Immunother, 2013.    36(2): p. 152-7.-   144. Shah, A. H., et al., Dendritic cell vaccine for recurrent    high-grade gliomas in pediatric and adult subjects: clinical trial    protocol. Neurosurgery, 2013. 73(5): p. 863-7.-   145. Reichardt, V.L., et al., Idiotype vaccination using dendritic    cells after autologous peripheral blood stem cell transplantation    for multiple myeloma-a feasibility study. Blood, 1999. 93(7): p.    2411-9.-   146. Lim, S. H. and R. Bailey-Wood, Idiotypic protein-pulsed    dendritic cell vaccination in multiple myeloma. Int J Cancer, 1999.    83(2): p. 215-22.-   147. Motta, M. R., et al., Generation of dendritic cells from CD14+    monocytes positively selected by immunomagnetic adsorption for    multiple myeloma patients enrolled in a clinical trial of    anti-idiotype vaccination. Br J Haematol, 2003. 121(2): p. 240-50.-   148. Reichardt, V. L., et al., Idiotype vaccination of multiple    myeloma patients using monocyte-derived dendritic cells.    Haematologica, 2003. 88(10): p. 1139-49.-   149. Guardino, A. E., et al., Production of myeloid dendritic    cells (DC) pulsed with tumor-specific idiotype protein for    vaccination of patients with multiple myeloma. Cytotherapy, 2006.    8(3): p. 277-89.-   150. Lacy, M. Q., et al., Idiotype-pulsed antigen-presenting cells    following autologous transplantation for multiple myeloma may be    associated with prolonged survival. Am J Hematol, 2009. 84(12): p.    799-802.-   151. Yi, Q., et al., Optimizing dendritic cell-based immunotherapy    in multiple myeloma: intranodal injections of idiotype-pulsed CD40    ligand-matured vaccines led to induction of type-1 and cytotoxic    T-cell immune responses in patients. Br J Haematol, 2010. 150(5): p.    554-64.-   152. Rollig, C., et al., Induction of cellular immune responses in    patients with stage-I multiple myeloma after vaccination with    autologous idiotype-pulsed dendritic cells. J Immunother, 2011.    34(1): p. 1006.-   153. Zahradova, L., et al., Efficacy and safety of Id-protein-loaded    dendritic cell vaccine in patients with multiple myeloma—phase II    study results. Neoplasma, 2012. 59(4): p. 440-9.-   154. Timmerman, J. M., et al., Idiotype-pulsed dendritic cell    vaccination for B-cell lymphoma: clinical and immune responses in 35    patients. Blood, 2002. 99(5): p. 1517-26.-   155. Maier, T., et al., Vaccination of patients with cutaneous    T-cell lymphoma using intranodal injection of autologous    tumor-lysate-pulsed dendritic cells. Blood, 2003. 102(7): p.    2338-44.-   156. Di Nicola, M., et al., Vaccination with autologous tumor-loaded    dendritic cells induces clinical and immunologic responses in    indolent B-cell lymphoma patients with relapsed and measurable    disease: a pilot study. Blood, 2009. 113(1): p. 18-27.-   157. Hus, I., et al., Allogeneic dendritic cells pulsed with tumor    lysates or apoptotic bodies as immunotherapy for patients with    early-stage B-cell chronic lymphocytic leukemia. Leukemia, 2005.    19(9): p. 1621-7.-   158. Li, L., et al., Immunotherapy for patients with acute myeloid    leukemia using autologous dendritic cells generated from leukemic    blasts. Int J Oncol, 2006. 28(4): p. 855-61.-   159. Roddie, H., et al., Phase I/II study of vaccination with    dendritic-like leukaemia cells for the immunotherapy of acute    myeloid leukaemia. Br J Haematol, 2006. 133(2): p. 152-7.-   160. Litzow, M. R., et al., Testing the safely of clinical-grade    mature autologous myeloid DC in a phase I clinical immunotherapy    trial of CML. Cytotherapy, 2006. 8(3): p. 290-8.-   161. Westermann, J., et al., Vaccination with autologous    non-irradiated dendritic cells in patients with bcr/abl+ chronic    myeloid leukaemia. Br J Haematol, 2007. 137(4): p. 297-306.-   162. Hus, I., et al., Vaccination of B-CLL patients with autologous    dendritic cells can change the frequency of leukemia    antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory    T cells toward an antileukemia response. Leukemia, 2008. 22(5): p.    1007-17.-   163. Palma, M., et al., Development of a dendritic cell-based    vaccine for chronic lymphocytic leukemia. Cancer Immunol    Immunother, 2008. 57(11): p. 1705-10.-   164. Van Tendeloo, V. F., et al.,Induction of complete and molecular    remissions in acute myeloid leukemia by Wilms' tumor 1    antigen-targeted dendritic cell vaccination. Proc Natl Acad Sci    USA, 2010. 107(31): p. 13824-9.-   165. Iwashita, Y., et al., A phase I study of autologous dendritic    cell-based immunotherapy for patients with unresectable primary    liver cancer. Cancer Immunol Immunother, 2003. 52(3): p. 155-61.-   166. Lee, W. C., et al., Vaccination of advanced hepatocellular    carcinoma patients with tumor lysate-pulsed dendritic cells: a    clinical trial. J Immunother, 2005. 28(5): p. 496-504.-   167. Butterfield, L. H., et al., A phase I/II trial testing    immunization of hepatocellular carcinoma patients with dendritic    cells pulsed with four alpha-fetoprotein peptides. Clin Cancer    Res, 2006. 12(9): p. 2817-25.-   168. Palmer, D. H., et al., A phase II study of adoptive    immunotherapy using dendritic cells pulsed with tumor lysate in    patients with hepatocellular carcinoma. Hepatology, 2009. 49(1): p.    124-32.-   169. El Ansary, M., et al., Immunotherapy by autologous dendritic    cell vaccine in patients with advanced HCC. J Cancer Res Clin    Oncol, 2013. 139(1): p. 39-48.-   170. Tada, F., et al., Phase I/II study of immunotherapy using tumor    antigen-pulsed dendritic cells in patients with hepatocellular    carcinoma. Int J Oncol, 2012. 41(5): p. 1601-9.-   171. Ueda, Y., et al., Dendritic cell-based inzmunotherapy of cancer    with carcinoembryonic antigen-derived, HLA-A24-restricted CTL    epitope: Clinical outcomes of 18 patients with metastatic    gastrointestinal or lung adenocarcinomas. Int J Oncol, 2004.    24(4): p. 909-17.-   172. Hirschowitz, E. A., et al., Autologous dendritic cell vaccines    for non-small-cell lung cancer. J Clin Oncol, 2004. 22(14): p.    2808-15.-   173. Chang, G. C., et al., A pilot clinical trial of vaccination    with dendritic cells pulsed with autologous tumor cells derived from    malignant pleural effusion in patients with late-stage lung    carcinoma. Cancer, 2005. 103(4): p. 763-71.-   174. Yannelli, J. R., et al., The large scale generation of    dendritic cells for the immunization of patients with non-small cell    lung cancer (NSCLC). Lung Cancer, 2005. 47(3): p. 337-50.-   175. Ishikawa, A., et al., A phase I study of    alpha-galactosylceramide (KRN7000)-pulsed dendritic cells in    patients with advanced and recurrent non-small cell lung cancer.    Clin Cancer Res, 2005. 11(5): p. 1910-7.-   176. Antonia, S. J., et al., Combination of p53 cancer vaccine with    chemotherapy in patients with extensive stage small cell lung    cancer. Clin Cancer Res, 2006. 12(3 Pt 1): p. 878-87.-   177. Perrot, I., et al., Dendritic cells infiltrating human    non-small cell lung cancer are blocked at immature stage. J    Immunol, 2007. 178(5): p. 2763-9.-   178. Hirschowitz, E. A., et al., Immunization of NSCLC patients with    antigen-pulsed immature autologous dendritic cells. Lung    Cancer, 2007. 57(3): p. 365-72.-   179. Baratelli, F., et al., Pre-clinical characterization of GMP    grade CCL21-gene modified dendritic cells for application in a phase    I trial in non-small cell lung cancer. J Transl Med, 2008. 6: p. 38.-   180. Hegmans, J. P., et al., Consolidative dendritic cell-based    immunotherapy elicits cytotoxicity against malignant mesothelioma.    Am J Respir Crit Care Med, 2010. 181(12): p. 1383-90.-   181. Urn, S. J., et al., Phase I study of autologous dendritic cell    tumor vaccine in patients with non-small cell lung cancer. Lung    Cancer, 2010. 70(2): p. 188-94.-   182. Chiappori, A. A., et al., INGN-225: a dendritic cell-based p53    vaccine (Ad.p53-DC) in small cell lung cancer: observed association    between immune response and enhanced chemotherapy effect. Expert    Opin Biol Ther, 2010. 10(6): p. 983-91.-   183. Perroud, M. W., Jr., et al., Mature autologous dendritic cell    vaccines in advanced non-small cell lung cancer: a phase I pilot    study. J Exp Clin Cancer Res, 2011. 30: p. 65.-   184. Skachkova, O. V., et al., Immunological markers of anti-tumor    dendritic cells vaccine efficiency in patients with non-mall cell    lung cancer. Exp Oncol, 2013. 35(2): p. 109-13.-   185. Hernando, J. J., et al., Vaccination with autologous tumour    antigen-pulsed dendritic cells in advanced gynaecological    malignancies: clinical and immunological evaluation of a phase I    trial. Cancer Immunol Immunother, 2002. 51(1): p. 45-52.-   186. Rahma, O. E., et al., A gynecologic oncology group phase II    trial of two p.53 peptide vaccine approaches: subcutaneous injection    and intravenous pulsed dendritic cells in high recurrence risk    ovarian cancer patients. Cancer Immunol Immunother, 2012. 61(3): p.    373-84.-   187. Chu, C. S., et al., Phase I/II randomized trial of dendritic    cell vaccination with or without cyclophosphamide for consolidation    therapy of advanced ovarian cancer in first or second remission.    Cancer Immunol Immunother, 2012. 61(5): p. 629-41.-   188. Kandalaft, L. E., et al., A Phase I vaccine trial using    dendritic cells pulsed with autologous oxidized lysate for recurrent    ovarian cancer. J Transl Med, 2013. 11: p. 149.-   189. Lepisto, A. J., et al., A phase I/II study of a MUC1 peptide    pulsed autologous dendritic cell vaccine as adjuvant therapy in    patients with resected pancreatic and biliary tumors. Cancer    Ther, 2008. 6(B): p.955-964.-   190. Rong, Y., et al., A phase I pilot trial of MUC1-peptide-pulsed    dendritic cells in the treatment of advanced pancreatic cancer. Clin    Exp Med, 2012. 12(3): p. 173-80.-   191. Endo, H., et al., Phase I trial of preoperative intratunzoral    injection of immature dendritic cells and OK-432 for resectable    pancreatic cancer patients. J Hepatobiliary Pancreat Sci, 2012.    19(4): p. 465-75.-   192. Tsujitani, S., et al., Infiltration of dendritic cells in    relation to tumor invasion and lymph node metastasis in human    gastric cancer. Cancer, 1990. 66(9): p. 2012-6.-   193. Tsujitani, S., et al., Postoperative adjuvant    immunochemotherapy and infiltration of dendritic cells for patients    with advanced gastric cancer. Anticancer Res, 1992. 12(3): p. 645-8.-   194. Kakeji, Y., et al., Prognostic significance of tumor-host    interaction in clinical gastric cancer: relationship between DNA    ploidy and dendritic cell infiltration. J Surg Oncol, 1993.    52(4): p. 207-12.-   195. Ishigami, S., et al., Prognostic value of HLA-DR expression and    dendritic cell infiltration in gastric cancer. Oncology, 1998. 55    (1): p. 65-9.-   196. Giannini, A., et al., Prognostic significance of accessory    cells and lymphocytes in nasopharyngeal carcinoma. Pathol Res    Pract, 1991. 187(4): p. 496-502.-   197. Gallo, O., et al., Correlations between histopathological and    biological findings in nasopharyngeal carcinoma and its prognostic    significance. Laryngoscope, 1991. 101(5): p. 487-93.-   198. Furihata, M., et al., HLA-DR antigen-and S-100 protein-positive    dendritic cells in esophageal squamous cell carcinoma—their    distribution in relation to prognosis. Virchows Arch B Cell Pathol    Incl Mol Pathol, 1992. 61(6): p. 409-14.-   199. Reichert, T. E., et al., The number of intratuntoral dendritic    cells and zeta-chain expression in T cells as prognostic and    survival biomarkers in patients with oral carcinoma. Cancer, 2001.    91(11): p. 2136-47.-   200. Timm, J., et al., Neoadjuvant immunotherapy of oral squamous    cell carcinoma modulates intratumoral CD4/CD8 ratio and tumor    microenvironment: a multicenter phase II clinical trial. J Clin    Oncol, 2005. 23(15): p. 3421-32.-   201. Nakano, T., et al., Antitumor activity of Langerhans cells in    radiation therapy for cervical cancer and its modulation with SPG    administration. In Vivo, 1993. 7(3): p. 257-63.-   202. Lespagnard, L., et al., Tumor-infiltrating dendritic cells in    adenocarcinomas of the breast: a study of 143 neoplasms with a    correlation to usual prognostic factors and to clinical outcome. Int    J Cancer, 1999. 84(3): p. 309-14.-   203. Iwamoto, M., et al., Prognostic value of tumor-infiltrating    dendritic cells expressing CD83 in human breast carcinomas. Int J    Cancer, 2003. 104(1): p. 92-7.-   204. Coventry, B. J. and J. Morton, CD1a-positive    infiltrating-dendritic cell density and 5-year survival from human    breast cancer. Br J Cancer, 2003. 89(3): p. 533-8.-   205. Zhao, R., et al., [Study on the relationship between the    dendritic cell infiltration in cancer tissues and prognosis in    patients with lung cancer]. Zhongguo Fei Ai Za Zhi, 2002. 5(2): p.    112-4.-   206. Diederichsen, A. C., et al., Prognostic value of the CD4+/CD8+    ratio of tumour infiltrating lymphocytes in colorectal cancer and    HLA-DR expression on tumour cells. Cancer Immunol Immunother, 2003.    52(7): p. 423-8.-   207. Dadabayev, A. R., et al., Dendritic cells in colorectal cancer    correlate with other tumor-infiltrating immune cells. Cancer Immunol    Immunother, 2004. 53(11): p. 978-86.-   208. Sandel, M. H., et al., Prognostic value of tumor-infiltrating    dendritic cells in colorectal cancer: role of maturation status and    intratumoral localization. Clin Cancer Res, 2005. 11(7): p. 2576-82.-   209. Yin, X. Y., et al., Prognostic significances of    tumor-infiltrating S-100 positive dendritic cells and lymphocytes in    patients with hepatocellular carcinoma.    Hepatogastroenterology, 2003. 50(53): p. 1281-4.-   210. Cai, X. Y., et al., Dendritic cell infiltration and prognosis    of human hepatocellular carcinoma. J Cancer Res Clin Oncol, 2006.    132(5): p. 293-301.-   211. Nakakubo, Y., et al., Clinical significance of immune cell    infiltration within gallbladder cancer. Br J Cancer, 2003. 89(9): p.    1736-42.-   212. Furihata, M., et al., Prognostic significance of CD83 positive,    mature dendritic cells in the gallbladder carcinoma. Oncol    Rep, 2005. 14(2): p. 353-6.-   213. Fukunaga, A., et al., CD8+ tumor-infiltrating lymphocytes    together with CD4+ tumor-infiltrating lymphocytes and dendritic    cells improve the prognosis of patients with pancreatic    adenocarcinoma. Pancreas, 2004. 28(1): p. e26-31.

1-18. (canceled)
 19. A method of enhancing an efficacy of an antibodyadministered to a patient, comprising the steps of: (a) administering anantibody capable of suppressing activity of a PD-L2 molecule; and (b)extracorporeally removing an immunological blocking factor that inhibitsthe effectiveness of the antibody from blood or a blood component of thepatient.
 20. The method of claim 19, wherein the antibody is selectedfrom the group consisting of AMP-224 and rHIgM12B7.
 21. The method ofclaim 19, wherein the immunological blocking factor is a soluble tumornecrosis factor (TNF)-alpha receptor.
 22. The method of claim 21,wherein step (b) is performed using an affinity capture substrate thatcomprises an immobilized TNF-alpha molecule comprising one or more of anative TNF-alpha molecule and a mutated TNF-alpha molecule.
 23. Themethod of claim 22, wherein the immobilized TNF-alpha molecule is atrimer.
 24. The method of claim 19, further comprising after step (b)administering an antibody capable of suppressing activity of a PD-L2molecule one or more additional times to the patient.
 25. The method ofclaim 19, further comprising after step (b) extracorporeally removingone or more additional times an immunological blocking factor thatinhibits the effectiveness of the antibody from blood or a bloodcomponent of the patient.
 26. The method of claim 19, wherein step (a)is performed before step (b).
 27. The method of claim 19, wherein step(b) is performed before step (a).
 28. An extracorporeal system forenhancing an efficacy of an antibody administered to a patient,comprising: a column configured to deplete a portion of an immunologicalblocking factor that reduces the efficacy of the antibody from blood ora blood component of the patient; wherein the antibody is capable ofsuppressing activity of a PD-L2 molecule.
 29. The system of claim 28,wherein the antibody is selected from the group consisting of AMP-224and rHIigM12B7.
 30. The system of claim 28, wherein the immunologicalblocking factor is a soluble tumor necrosis factor (TNF)-alpha receptor.31. The system of claim 30, wherein the column comprises an affinitycapture substrate that comprises an immobilized TNF-alpha moleculecomprising one or more of a native TNF-alpha molecule or a mutatedTNF-alpha molecule.
 32. The system of claim 31, wherein the affinitycapture substrate is a trimer.